# Rapid point-of-care detection of Hepatitis C viral RNA using multiplexed CRISPR/Cas platforms

> **NIH NIH R21** · UNIVERSITY OF FLORIDA · 2023 · $228,750

## Abstract

PROJECT ABSTRACT/SUMMARY
 In 2017, WHO estimated 71 million people had chronic Hepatitis C Virus (HCV) but 40-50% of the living
patients were unaware of their infection status. In 2016 alone, an estimated 399,000 HCV-related deaths were
reported by WHO. CDC estimates that between 2013-2016, around 2.4 million people were infected with HCV
within the US and only a fraction of them were diagnosed properly. A rapid and inexpensive detection of HCV
RNA would allow quicker intervention and can significantly reduce the risk of death and infection rate, especially
in resource-limited settings. By engineering and multiplexing CRISPR/Cas systems in unique ways ultra-
sensitive detection of low copies of HCV RNA can be achieved for blood samples within 30 minutes. This project
proposes development and clinical validation of two highly innovative CRISPR-based approaches for detecting
HCV genotype in a lateral flow assay. Type V and VI CRISPR/Cas systems when bound with their specific target
nucleic acid sequence, activate a secondary collateral nuclease activity that can rapidly cleave single-stranded
nucleic acids in a non-specific multiple turnover manner. This collateral nuclease activity has been utilized for
rapidly detecting nucleic acids. However, they have nanomolar sensitivity and require pre-amplification of a target
to achieve attomolar detection that is desirable for clinical use. While pre-amplification can be achieved by
isothermal techniques this requires additional manipulation steps and a stable temperature control increasing
the time and cost of an assay.
 To eliminate the need for target pre-amplification while maintaining high sensitivity and specificity, this
high-risk/high-reward project proposes two innovative approaches to achieve rapid detection of HCV RNA
without any target amplification. For the first aim, a recently developed `CRISPR-ENHANCE' (CE) platform from
the PI's lab that achieved femtomolar detection of nucleic acids in 30 minutes without any target pre-amplification
(Nguyen et al., Nat. Comm., 2020) will be tested in a combinatorial fashion to further enhance the sensitivity for
detecting clinical levels of HCV RNA using a lateral flow assay. The second aim is to develop and clinically
validate a CRISPR Chain Reaction (CCR) based test for detecting HCV RNA and genotypes using multiplexed
lateral flow assay and quantifying RNA using a simple fluorescence-based point-of-care device. Both the
approaches will be clinically validated in banked samples with acute/chronic infections as well as longitudinally
monitor patients undergoing anti-viral therapy in collaboration with Hepatitis C Therapeutic Registry and
Research Network (HCV-TARGET), an international consortium of leading HCV investigators. All the
components as defined by the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust,
Equipment-free and Deliverable to end-users) criteria by WHO. The development of a rapid diagnostic platform
would allow...

## Key facts

- **NIH application ID:** 10613983
- **Project number:** 5R21AI168795-02
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Piyush K Jain
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $228,750
- **Award type:** 5
- **Project period:** 2022-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10613983

## Citation

> US National Institutes of Health, RePORTER application 10613983, Rapid point-of-care detection of Hepatitis C viral RNA using multiplexed CRISPR/Cas platforms (5R21AI168795-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10613983. Licensed CC0.

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